: This grant application is focused on elucidating the molecular basis of clinical resistance to HIV-1 PR inhibitors. Resistant mutants of HIV-1 PR show lower affinity for inhibitors, yet are still able to process viral polyprotein substrates in the course of viral replication. The amino acid sequences of resistant mutants are known from clinical studies. In order to elucidate the molecular basis of resistance to HIV-1 protease inhibitors, the complete binding energetics of substrate and inhibitors to the HIV-1 PR (wt; resistance mutants) will be measured by high sensitivity reaction calorimetric techniques and standard spectroscopic assays. Structure-based thermodynamic analysis will be performed using a novel approach developed in the applicant's laboratory. This information will be used to develop a structural map of the binding energetics of substrates and inhibitors. The structural map will identify the energetic contributions from each group in the protease, substrate or inhibitor molecules to the overall binding affinity. This map will also identify enthalpic and entropic contributions to the binding energetics, and dissect the entropy contributions into conformation and solvent-related components. Differences in the type and strength of interactions will be evaluated and used to identify why some specific mutations affect the binding affinity of the substrate and inhibitor in different ways.